Journal: Human Molecular Genetics

Article Title: Amyotrophic lateral sclerosis-associated TDP-43 mutation Q331K prevents nuclear translocation of XRCC4-DNA ligase 4 complex and is linked to genome damage-mediated neuronal apoptosis

doi: 10.1093/hmg/ddz062

Figure Lengend Snippet: Targeted sequencing of exon-6 of the TARDBP gene in spinal cord specimens from ALS patients. (A) Chromatogram showing detection of the Q331K mutation in genomic DNA isolated from spinal cord tissue from an ALS patient. (B) Immunoblots probed with TDP-43 antibody and γH2AX antibody show reduced monomeric TDP-43 protein and increased γH2AX in an ALS specimen carrying the Q331K mutation compared to an age-matched control sample. GAPDH served as the loading control. (C) Analysis of DNA integrity by LA-PCR. Representative agarose gel images indicate reduced DNA amplification of OCT3/4, NONOG, Polβ and HPRT gene segments in TDP-43-Q331K spinal cord compared to control. The 200 bp short amplicon served as control.( D) Quantitation of PCR products by Pico Green-based DNA quantitation from triplicate experiments. (E) IHC of spinal cord from an ALS patient carrying a Q331K mutation for TDP-43, γH2AX and TUNEL staining. Representative images acquired at 20× magnification. (F) Quantitation of mean γH2AX signal and mean TUNEL-positive cells per field in ALS spinal cord sample and age-matched controls. **P<0.05; ****P<0.0001.

Article Snippet: The TDP-43 coding DNA sequence (CDS) along with the N-terminal 1X FLAG tag sequence was PCR-amplified from FLAG-TDP-43 pcDNA 3.1 vector using high-fidelity Deep Vent DNA polymerase (# M0258, NEB, Ipswich, MA).

Techniques: Sequencing, Mutagenesis, Isolation, Western Blot, Agarose Gel Electrophoresis, Amplification, Quantitation Assay, TUNEL Assay, Staining

Journal: Human Molecular Genetics

Article Title: Amyotrophic lateral sclerosis-associated TDP-43 mutation Q331K prevents nuclear translocation of XRCC4-DNA ligase 4 complex and is linked to genome damage-mediated neuronal apoptosis

doi: 10.1093/hmg/ddz062

Figure Lengend Snippet: Q331K expression induces ROS stress and accumulation DNA strand breaks in neurons. (A) Cellular Reactive Oxygen Species Detection Assay by IF microscopy. Upper panels represent WT-expressing cells; lower panels represent Q331K-expressing cells. The two panels on the left represent uninduced cells. Panels to the right represent cells after Dox induction. Deep RED dye staining indicates the presence of ROS (Scale bar, 10 μm). (B) Quantitation of cellular ROS using a microplate fluorescence reader. (C) LA-PCR analysis of genomic DNA isolated from TDP-43-Q331K neurons shows reduced DNA integrity. Representative agarose gel images of amplified DNA products. (D) Quantification of PCR products by Pico Green-based DNA quantitation from triplicate experiments. (E) Alkaline comet analysis of differentiated SH-SY5Y cells expressing WT or Q331K (lower panel). Quantitation of mean tail moment before and after Dox induction in 25–50 cells reveals an ~5-fold increase in DNA damage in Q331K cells (Scale bar, 10 μm). *P<0.01; **P<0.05; ***P<0.0005.

Article Snippet: The TDP-43 coding DNA sequence (CDS) along with the N-terminal 1X FLAG tag sequence was PCR-amplified from FLAG-TDP-43 pcDNA 3.1 vector using high-fidelity Deep Vent DNA polymerase (# M0258, NEB, Ipswich, MA).

Techniques: Expressing, Detection Assay, Microscopy, Staining, Quantitation Assay, Fluorescence, Isolation, Agarose Gel Electrophoresis, Amplification

Journal: Human Molecular Genetics

Article Title: Amyotrophic lateral sclerosis-associated TDP-43 mutation Q331K prevents nuclear translocation of XRCC4-DNA ligase 4 complex and is linked to genome damage-mediated neuronal apoptosis

doi: 10.1093/hmg/ddz062

Figure Lengend Snippet: The Q331K mutation affects the nuclear translocation of XRCC4-DNA ligase 4. (A) IF of DNA ligase 4 in WT or Q331K cells shows increased cytoplasmic presence in mutant cells (Scale bar, 10 μm). The 2.5-dimensional view of the localization is shown in the image below. (B) IF of XRCC4 in WT or Q331K cells shows their reduced nuclear presence in mutant cells (Scale bar, 10 μm). The 2.5-dimensional view of the localization is shown in the image below. (C) PLA of FLAG versus DNA ligase 4 and FLAG versus XRCC4 in cells expressing WT and mutant TDP-43 (Scale bar, 10 μm). The higher number of foci in the cytoplasm of Q331K-expressing cells compared to WT-expressing cells indicates the increased interaction of XRCC4-DNA ligase 4 after DNA-damage induction by IR (3 Gy). **P<0.05 ; ****P<0.0001.

Article Snippet: The TDP-43 coding DNA sequence (CDS) along with the N-terminal 1X FLAG tag sequence was PCR-amplified from FLAG-TDP-43 pcDNA 3.1 vector using high-fidelity Deep Vent DNA polymerase (# M0258, NEB, Ipswich, MA).

Techniques: Mutagenesis, Translocation Assay, Expressing

Journal: Human Molecular Genetics

Article Title: Amyotrophic lateral sclerosis-associated TDP-43 mutation Q331K prevents nuclear translocation of XRCC4-DNA ligase 4 complex and is linked to genome damage-mediated neuronal apoptosis

doi: 10.1093/hmg/ddz062

Figure Lengend Snippet: The Q331K mutation affects DNA-ligation activity. (A) In vitro DNA-ligation assay using extracts of neurons expressing WT or mutant TDP-43 before and after DNA damage induction by IR (3 Gy). Quantitation of DNA-ligation activity from three independent experiments expressed as fold change in the histogram. (B) IP of WT- and Q331K-expressing cells with anti-FLAG antibody or IgG (mouse) before and after DNA-damage induction by IR (3 Gy) shows reduce DNA ligase 4 interaction in Q331K mutant cells. (C) His-affinity pulldown assay using recombinant TDP-43-WT and TDP-43-Q331K shows reduced association of the Q331K protein with XRCC4-DNA ligase 4 complex in vitro. (D) A model showing TDP-43 WT facilitates XRCC4-Ligase4 complex nuclear translocation in healthy neuronal cells leading to efficient DNA repair. ALS-linked Q331K mutation nuclear clearance and cytoplasmic aggregation impairs XRCC4-Ligase4 complex nuclear translocation leading to persistent DNA damage accumulation. **P<0.05.

Article Snippet: The TDP-43 coding DNA sequence (CDS) along with the N-terminal 1X FLAG tag sequence was PCR-amplified from FLAG-TDP-43 pcDNA 3.1 vector using high-fidelity Deep Vent DNA polymerase (# M0258, NEB, Ipswich, MA).

Techniques: Mutagenesis, DNA Ligation, Activity Assay, In Vitro, Expressing, Quantitation Assay, Recombinant, Translocation Assay

Journal: Epigenetics

Article Title: SFRP1 CpG island methylation locus is associated with renal cell cancer susceptibility and disease recurrence.

doi: 10.4161/epi.19614

Figure Lengend Snippet: Figure 1. Quantitative SFRP1 CGI methylation and gene expression analysis in renal cell lines and normal primary cells. (A) Illustration of SFRP1 CGI structure and relative positions of methylation analysis. (B) Example for quantitative methylation analysis of 11 CpG sites (gray bars) and one control site (narrow gray bar) in the CGI region of the SFRP1 gene using pyrosequencing of bisulfite treated DNA. Here, an average methylation of 35% was determined while a nearly complete bisulfite conversion of 99.6% has been achieved. (C) Quantitative SFRP1 CGI methylation analysis and relative quantitation of SFRP1 mRNA expression in renal cell cancer cell lines and primary cells from normal kidney (RPTEC). (D) Duplicate measurements and Lin’s concordance correlation analysis. The solid line represents the regression line of measurements while the dashed line indicates the line of perfect concordance. Only minute deviations from ideal concordance were observed.

Article Snippet: Colella S, Shen L, Baggerly KA, Issa JP, Krahe R. Sensitive and quantitative universal Pyrosequencing methylation analysis of CpG sites.

Techniques: Methylation, Gene Expression, Control, Quantitation Assay, Expressing

Journal: Cancers

Article Title: Allelic Expression Imbalance Analysis Identified YAP1 Amplification in p53- Dependent Osteosarcoma

doi: 10.3390/cancers13061364

Figure Lengend Snippet: Genome-wide allelic expression imbalance analysis in osteosarcoma. ( A ) A Manhattan plot showing the result of the genome-wide allelic imbalance analysis. The most significant SNPs were found on pig chromosomes 6, 9, 14 and 16. ( B ) Schematic genomic structure of the YAP1-BIRC3 locus on chromosome 9 in pigs. The blue arrow indicates the position of the 9:33044172 A/G SNP in the 3′UTR of BIRC3 . ( C ) cDNA pyrosequencing result for the SNP 9:33044172 A/G in osteosarcoma (os, n = 48) and matched healthy bone (b) samples collected from flTP53 R167H pigs. To test analysis the validity of the pyrosequencing assay, we used DNA samples ( n = 5) extracted from wild-type pigs. *** p < 0.001.

Article Snippet: Sections were stained with biotinylated rabbit anti-YAP1 antibody (diluted 1:200; ARP50530_P050, Aviva System Biology Cooperation, San Diego, CA, USA) and binding visualized with the avidin-peroxidase solution (ABC kit, Vector, Darmstadt, Germany) followed by DAB staining (Vector).

Techniques: Genome Wide, Expressing, Pyrosequencing Assay

Journal: Cancers

Article Title: Allelic Expression Imbalance Analysis Identified YAP1 Amplification in p53- Dependent Osteosarcoma

doi: 10.3390/cancers13061364

Figure Lengend Snippet: YAP1 amplification in p53 deficient osteosarcoma. ( A ) Point plot showing the correlation between 9:33044172 A allele expression and YAP1 copy number. Gray and red points show expression of A allele in bone and OS samples, respectively. Blue points show the measurements in wild-type samples. ( B ) Point plot showing the correlation between 9:33044172 A allele expression and OS ( n = 48) size. ( C ) Point plot showing the correlation between YAP1 copy number and OS ( n = 48) size. ( D ) Quantitative PCR of YAP1 mRNA expression in wild type (wt, n = 5) bones, as well as OS ( n = 48) and matched healthy bone samples from flTP53 R167H pigs. ( E ) Representative Western blot showing YAP1 expression in wild type bone, OS and healthy bone samples from flTP53 R167H pigs. The uncropped Western blots have been shown in . ( F ) Immunohistochemistry staining showing the nuclear location of YAP1 in sections of osteosarcoma from flTP53 R167H pigs. Control samples show staining without the first antibody. Scale bars- 100 μm. (** p < 0.01)

Article Snippet: Sections were stained with biotinylated rabbit anti-YAP1 antibody (diluted 1:200; ARP50530_P050, Aviva System Biology Cooperation, San Diego, CA, USA) and binding visualized with the avidin-peroxidase solution (ABC kit, Vector, Darmstadt, Germany) followed by DAB staining (Vector).

Techniques: Amplification, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Staining, Control

Journal: Cancers

Article Title: Allelic Expression Imbalance Analysis Identified YAP1 Amplification in p53- Dependent Osteosarcoma

doi: 10.3390/cancers13061364

Figure Lengend Snippet: In vitro functional analysis of YAP1 deficiency in p53 deficient primary osteosarcoma cells. ( A ) Sequence analysis showing the result of CRISPR/Cas9 editing of YAP1 in pig OS cells. ( B ) Western blot showing the lack of YAP1 protein in the edited flTP53 R167H OS cells. ( C ) Representative microscopic view showing the morphology of YAP1 −/− /flTP53 R167H OS cells. As a control, flTP53 R167H OS cells were transfected with the GFP control vector (left scale bars, 400μm; right scale bars, 200 μm) ( D ) Proliferation result for YAP1 −/− /flTP53 R167H and flTP53 R167H OS cells. ( E ) Representative microscopic images showing a difference in migration and invasion between YAP1 −/− /flTP53 R167H and flTP53 R167H OS cells (scale bars, 200 μm). Quantitative measurement of migration ( F ) and invasion ( G ). ( H ) Immunofluorescence staining for Ki67 and DAPI in YAP1 −/− /flTP53 R167H and flTP53 R167H OS cells. ( I ) Quantification rates of the Ki67 positive cells. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Sections were stained with biotinylated rabbit anti-YAP1 antibody (diluted 1:200; ARP50530_P050, Aviva System Biology Cooperation, San Diego, CA, USA) and binding visualized with the avidin-peroxidase solution (ABC kit, Vector, Darmstadt, Germany) followed by DAB staining (Vector).

Techniques: In Vitro, Functional Assay, Sequencing, CRISPR, Western Blot, Control, Transfection, Plasmid Preparation, Migration, Immunofluorescence, Staining

Journal: Cancers

Article Title: Allelic Expression Imbalance Analysis Identified YAP1 Amplification in p53- Dependent Osteosarcoma

doi: 10.3390/cancers13061364

Figure Lengend Snippet: Expression of p53 related genes in YAP1 −/− /flTP53 R167H OS cells. ( A ) RT-PCR result for WRAP53, TP53INP1, p14, p16, RB1, TP63, TP73 in YAP1 −/− /flTP53 R167H and flTP53 R167H OS cells. Three independent transfections for each expression vector were performed. NC—negative control. ( B ) Quantitative RT-PCR of p16 mRNA expression. GAPDH mRNA expression was used as a reference. ** p < 0.01. ( C ) Western blot showing lack of p63 expression in YAP1 −/− /flTP53 R167H OS cells.

Article Snippet: Sections were stained with biotinylated rabbit anti-YAP1 antibody (diluted 1:200; ARP50530_P050, Aviva System Biology Cooperation, San Diego, CA, USA) and binding visualized with the avidin-peroxidase solution (ABC kit, Vector, Darmstadt, Germany) followed by DAB staining (Vector).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Negative Control, Quantitative RT-PCR, Western Blot

Journal: Cancers

Article Title: Allelic Expression Imbalance Analysis Identified YAP1 Amplification in p53- Dependent Osteosarcoma

doi: 10.3390/cancers13061364

Figure Lengend Snippet: DNA methylation analysis of the p16 and Rb1 promoter regions in YAP1 −/− /flTP53 R167H OS cells. ( A ) Pyrosequencing result at 8 CpG sites in the p16 promoter region in YAP1 −/− /flTP53 R167H ( n = 3) and flTP53 R167H ( n = 3) OS cells. ( B ) Pyrosequencing result at 9 CpG sites in the Rb1 promoter region in YAP1 −/− /flTP53 R167H ( n = 3) and flTP53 R167H ( n = 3) OS cells. * p < 0.05.

Article Snippet: Sections were stained with biotinylated rabbit anti-YAP1 antibody (diluted 1:200; ARP50530_P050, Aviva System Biology Cooperation, San Diego, CA, USA) and binding visualized with the avidin-peroxidase solution (ABC kit, Vector, Darmstadt, Germany) followed by DAB staining (Vector).

Techniques: DNA Methylation Assay